A Real-Time PCR/SYBR Green I Method for the Rapid Quantification of Salmonella enterica in Poultry Meat
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A Real-Time PCR/SYBR Green I Method for the Rapid Quantification of Salmonella enterica in Poultry Meat Caterina Agrimonti & Laura Bortolazzi & Elena Maestri & Anna Maria Sanangelantoni & Nelson Marmiroli
Received: 7 August 2012 / Accepted: 30 January 2013 / Published online: 10 March 2013 # Springer Science+Business Media New York 2013
Abstract It has been developed a method for quantitative detection of Salmonella enterica in poultry meat based on real-time PCR (qtPCR) with species-specific primers and SYBR® GreenER™ chemistry. Two methods for bacterial DNA extraction were compared: one based on a commercial kit (AccuPrep®) and the other on silica–magnetite nanoparticles. Primers were designed on sequence of invA gene encoding for an inner membrane protein associated with invasiveness of Salmonella. Serial dilutions of DNA from pure cultures of Salmonella and from broiler breast samples spiked with serial dilutions of Salmonella were analyzed in different replicates and with different PCR equipments. Robustness of the method was evaluated and compared in terms of repeatability, reproducibility, and consistency with conventional plate count methods and for applicability to the different equipments. The matrix effect upon each reaction specificity was assessed with addition of DNA from a noncompetitive internal amplification control. The limit of detection (LOD) was determined between 10 and 40 colonyforming units (CFUs)/ml; whereas, the limit of quantification (LOQ) was 102 CFUs/ml. Quantification with qtPCR was in the same order of magnitude as enumeration with plate counting but with an overestimation. Keywords Bacteria enumeration . Poultry food chain . Quantitative real-time PCR . Food safety . DNA extraction
Electronic supplementary material The online version of this article (doi:10.1007/s12161-013-9583-y) contains supplementary material, which is available to authorized users. C. Agrimonti : L. Bortolazzi : E. Maestri : A. M. Sanangelantoni : N. Marmiroli (*) Department of Life Sciences, University of Parma, Viale Parco Area delle Scienze 11/A, 43124 Parma, Italy e-mail: [email protected]
Introduction Salmonella is one of the most important pathogens, within the family of Enterobacteriaceae, a common cause in foodborne disease (Darwin and Miller 1999; European Food Safety Authority 2007; Foley and Lynne 2008). Nowadays, it is generally accepted that there are only two species of Salmonella: Salmonella enterica and Salmonella bongori (Brenner et al. 2000) though over 2,500 serovars responsible for zoonosis were reported. The European Food Safety Authority (2009) identified Salmonella as the most frequent cause of illness, with 2,201 outbreaks in 22 EU member states, affecting over 8,900 people. Recipients of Salmonella are pork, eggs, and poultry, the chicks being extremely susceptible to infections with S. enterica subspecies (Darwin and Miller 1999; Tauxe 2002; Berndt et al. 2007). In humans, Salmonella causes different diseases including gastroenteritis, bacteremia, and typhoid fevers (Foley and
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